Description: Objectives To investigate the pathological and clinical meaning of p63 in extranodal nasal type NK/T cell lymphoma (NKTCL). Methods We detected p63 and p53 expression using immunohistochemistry staining in 84 cases of NKTCL from Southern of China, an area with a well known high incidence of nasopharyngeal carcinoma, which is closely associated with Epstein–Barr virus infection. Moreover, we analysed the relationship between p63 and p53 expression and the clinicopathological features of NKTCL. Results Our results first showed that p63 expression was found in 14.3% (12/84) of NKTCL compared with 6.6% (2/30) in reactive lymphoid hyperplasia of nasopharynx. p63 Expression rate in NKTCL was significantly higher than that in reactive lymphoid hyperplasia of nasopharynx (p=0.016). NKTCL patients with p63 positivity had poorer 5-year overall survival rate (29.2%) than that (49.9%) of p63 negativity. p53 expression was found in 33.3% (28/84) of NKTCL. Our data showed that p53 expression was significantly associated with tumour stage (p=0.016) and international prognostic index (p=0.026) in patients with NKTCL. Cox regression test showed that p53 expression rate and international prognostic index score were statistically independent prognostic factors for NKTCL patients (p=0.002 and p=0.016, respectively). Our results suggest that p63 and p53 might play a role in pathogenesis of NKTCL.

Description: Background Mounting epidemiological evidence supports a role for phosphatase and tensin homologue (PTEN)-T cell leukaemia 1 (Tcl1) signalling deregulation in hepatocarcinogenesis. Objective To determine the molecular and biochemical mechanisms by which the PTEN/Tcl1 axis regulates the pentose phosphate pathway (PPP) in hepatocellular carcinoma (HCC). Methods We compared levels of PTEN and glucose-6-phosphate dehydrogenase (G6PD) mRNA in human HCC and healthy liver tissue. We measured PPP flux, glucose consumption, lactate production, nicotinamide adenine dinucleotide phosphate (NADPH) levels and lipid accumulation. We investigated the PTEN/Tcl1 axis using molecular biology, biochemistry and mass spectrometry analysis. We assessed proliferation, apoptosis and senescence in cultured cells, and tumour formation in mice. Results We showed that PTEN inhibited the PPP pathway in human liver tumours. Through the PPP, PTEN suppressed glucose consumption and biosynthesis. Mechanistically, the PTEN protein bound to G6PD, the first and rate-limiting enzyme of the PPP and prevented the formation of the active G6PD dimer. Tcl1, a coactivator for Akt, reversed the effects of PTEN on biosynthesis. Tcl1 promoted G6PD activity and also increased G6PD pre-mRNA splicing and protein expression in a heterogeneous nuclear ribonucleoprotein (hnRNPK)-dependent manner. PTEN also formed a complex with hnRNPK, which inhibited G6PD pre-mRNA splicing. Moreover, PTEN inactivated Tcl1 via glycogen synthase kinase-3β (GSK3β)-mediated phosphorylation. Importantly, Tcl1 knockdown enhanced the sensitivity of HCC to sorafenib, whereas G6PD knockdown inhibited hepatocarcinogenesis. Conclusions These results establish the counteraction between PTEN and Tcl1 as a key mechanism that regulates the PPP and suggest that targeting the PTEN/Tcl1/hnRNPK/G6PD axis could open up possibilities for therapeutic intervention and improve the prognosis of patients with HCC.

Description: Nucleolin is a ubiquitously expressed protein and participates in many important biological processes, such as cell cycle regulation and ribosomal biogenesis. The activity of nucleolin is regulated by intracellular localization and post-translational modifications, including phosphorylation, methylation, and ADP-ribosylation. Small ubiquitin-like modifier (SUMO) is a category of recently verified forms of post-translational modifications and exerts various effects on the target proteins. In the studies reported here, we discovered SUMOylational modification of human nucleolin protein at Lys-294, which facilitated the mRNA binding property of nucleolin by maintaining its nuclear localization. In response to arsenic exposure, nucleolin-SUMO was induced and promoted its binding with gadd45α mRNA, which increased gadd45α mRNA stability and protein expression, subsequently causing GADD45α-mediated cell death. On the other hand, ectopic expression of Mn-SOD attenuated the arsenite-generated superoxide radical level, abrogated nucleolin-SUMO, and in turn inhibited arsenite-induced apoptosis by reducing GADD45α expression. Collectively, our results for the first time demonstrate that nucleolin-SUMO at K294R plays a critical role in its nucleus sequestration and gadd45α mRNA binding activity. This novel biological function of nucleolin is distinct from its conventional role as a proto-oncogene. Therefore, our findings here not only reveal a new modification of nucleolin protein and its novel functional paradigm in mRNA metabolism but also expand our understanding of the dichotomous roles of nucleolin in terms of cancer development, which are dependent on multiple intracellular conditions and consequently the appropriate regulations of its modifications, including SUMOylation.

Description: RING proteins constitute the largest class of E3 ubiquitin ligases. Unlike most RINGs, AO7 (RNF25) binds the E2 ubiquitin-conjugating enzyme, UbcH5B (UBE2D2), with strikingly high affinity. We have defined, by co-crystallization, the distinctive means by which AO7 binds UbcH5B. AO7 contains a structurally unique UbcH5B binding region (U5BR) that is connected by an 11-amino acid linker to its RING domain, forming a clamp surrounding the E2. The U5BR interacts extensively with a region of UbcH5B that is distinct from both the active site and the RING-interacting region, referred to as the backside of the E2. An apparent paradox is that the high-affinity binding of the AO7 clamp to UbcH5B, which is dependent on the U5BR, decreases the rate of ubiquitination. We establish that this is a consequence of blocking the stimulatory, non-covalent, binding of ubiquitin to the backside of UbcH5B. Interestingly, when non-covalent backside ubiquitin binding cannot occur, the AO7 clamp now enhances the rate of ubiquitination. The high-affinity binding of the AO7 clamp to UbcH5B has also allowed for the co-crystallization of previously described and functionally important RING mutants at the RING-E2 interface. We show that mutations having marked effects on function only minimally affect the intermolecular interactions between the AO7 RING and UbcH5B, establishing a high degree of complexity in activation through the RING-E2 interface.

Description: Objective miR-21 was found to be overexpressed in the colon tissues and serum of patients with UC and colorectal cancer (CRC); however, the exact roles of miR-21 in colitis-associated CRC remain unclear. The aim of our study was to investigate the biological mechanisms of miR-21 in colitis-associated colon cancer (CAC). Design miR-21 expression was examined in the tumours of 62 patients with CRC from China and 37 colitis-associated neoplastic tissues from Japan and Austria. The biological functions of miR-21 were studied using a series of in vitro, in vivo and clinical approaches. Results miR-21 levels were markedly upregulated in the tumours of 62 patients with CRC, 22 patients with CAC, and in a mouse model of CAC. Following azoxymethane and dextran sulfate sodium intervention, miR-21-knockout mice showed reduced expression of proinflammatory and procarcinogenic cytokines (interleukin (IL) 6, IL-23, IL-17A and IL-21) and a decrease in the size and number of tumours compared with the control mouse group. The absence of miR-21 resulted in the reduced expression of Ki67 and the attenuated proliferation of tumour cells with a simultaneous increase in E-cadherin and decrease in β-catenin and SOX9 in the tumours of CAC mice. Furthermore, the absence of miR-21 increased the expression of its target gene PDCD4 and subsequently modulated nuclear factor (NF)-B activation. Meanwhile, miR-21 loss reduced STAT3 and Bcl-2 activation, causing an increase in the apoptosis of tumour cells in CAC mice. Conclusions These observations provide novel evidence for miR-21 blockade to be a key strategy in reducing CAC.

Description: Purpose To investigate p53 and hepatoma-derived growth factor (HDGF) expression and their association with clinicopathological features of Ewing family tumour (EFT). Experimental design A total of 108 cases of EFT were retrospectively analysed. p53 and HDGF expression were detected using immunohistochemistry, and the relationships between p53 expression and HDGF expression and clinicopathological features of EFT were analysed. Kaplan–Meier curves were applied to estimate overall survival, log-rank test was used to assess prognostic relevance of p53 expression with overall survival and Cox regression model was performed to evaluate HRs. Results p53 expression and high HDGF expression was found in 17 (15.7%) and 55 (50.9%) patients, respectively. p53 expression was significantly associated with metastatic stage at initial diagnosis (p=0.007) and tumour venous/nerve invasion (p=0.023). A significant positive correlation was found between p53 expression and HDGF expression in EFT (p=0.022). p53 expression was an independent prognostic factor for overall survival of patients with EFT (p〈0.001). Patients with p53-positive/high HDGF expression had a significantly shorter overall survival than those with p53-positive/low HDGF expression or p53-negative/high HDGF expression or p53-negative/low HDGF expression. We first constructed a novel molecular staging system by combining p53 expression and HDGF expression, which significantly improved prognostic stratification for patients with EFT. Conclusions p53 expression was an independent prognostic factor for patients with EFT. Combining p53 expression and HDGF expression significantly improved prognostic stratification for patients with EFT.

Description: The application of functional genomic analysis of breast cancer metastasis has led to the identification of a growing number of organ-specific metastasis genes, which often function in concert to facilitate different steps of the metastatic cascade. However, the gene regulatory network that controls the expression of these metastasis genes remains largely unknown. Here, we demonstrate a computational approach for the deconvolution of transcriptional networks to discover master regulators of breast cancer bone metastasis. Several known regulators of breast cancer bone metastasis such as Smad4 and HIF1 were identified in our analysis. Experimental validation of the networks revealed BACH1, a basic leucine zipper transcription factor, as the common regulator of several functional metastasis genes, including MMP1 and CXCR4. Ectopic expression of BACH1 enhanced the malignance of breast cancer cells, and conversely, BACH1 knockdown significantly reduced bone metastasis. The expression of BACH1 and its target genes was linked to the higher risk of breast cancer recurrence in patients. This study established BACH1 as the master regulator of breast cancer bone metastasis and provided a paradigm to identify molecular determinants in complex pathological processes.

Description: Mature B cells (BCs) express CD23 and B cell receptors. Whether activation of CD23 and B cell receptors has different effects on BC activities is unclear. This study aims to investigate the mechanism by which the specific antigen immunotherapy regulates the activation of BCs in the skewed Th2 responses. Mice were sensitized to ovalbumin. The specific antigen vaccination (SAV) at graded doses was employed to modulate the activities of BCs in which the expression of IL-10, IgE, matrix metalloproteinase-9 (MMP9), CD23, and serum soluble CD23 by BCs was evaluated. The immune regulatory effect of BCs primed by lower or higher SAV doses was observed with an adoptive transfer mouse experiment. SAV activated CD23 to produce IL-10 in BCs at lower doses. The higher doses of SAV increased the expression of MMP9 in BCs that reduced the amounts of CD23 in BCs and increased the serum levels of soluble CD23, which was abrogated by the pretreatment with MMP9 inhibitor. Adoptively transfer with BCs primed by lower doses of SAV inhibited the ongoing antigen-specific Th2 responses whereas the BCs primed by higher doses of SAV exacerbated the ongoing Th2 responses. Exposure to specific antigens at optimal doses can activate BCs to produce IL-10 to suppress the skewed antigen-specific Th2 responses. The antigen doses of SAV higher than the optimal doses may promote the production of soluble CD23 to exacerbate the ongoing immune responses.

Description: A hallmark of severe Lassa fever is the generalized immune suppression, the mechanism of which is poorly understood. Lassa virus (LASV) nucleoprotein (NP) is the only known 3′-5′ exoribonuclease that can suppress type I interferon (IFN) production possibly by degrading immune-stimulatory RNAs. How this unique enzymatic activity of LASV NP recognizes and processes RNA substrates is unknown. We provide an atomic view of a catalytically active exoribonuclease domain of LASV NP (LASV NP-C) in the process of degrading a 5′ triphosphate double-stranded (ds) RNA substrate, a typical pathogen-associated molecular pattern molecule, to induce type I IFN production. Additionally, we provide for the first time a high-resolution crystal structure of an active exoribonuclease domain of Tacaribe arenavirus (TCRV) NP. Coupled with the in vitro enzymatic and cell-based interferon suppression assays, these structural analyses strongly support a unified model of an exoribonuclease-dependent IFN suppression mechanism shared by all known arenaviruses. New knowledge learned from these studies should aid the development of therapeutics against pathogenic arenaviruses that can infect hundreds of thousands of individuals and kill thousands annually.

Description: Toxin YafQ functions as a ribonuclease in the dinJ-yafQ toxin-antitoxin system of Escherichia coli. Antitoxin DinJ neutralizes YafQ-mediated toxicity by forming a stable protein complex. Here, crystal structures of the (DinJ)2-(YafQ)2 complex and the isolated YafQ toxin have been determined. The structure of the heterotetrameric complex (DinJ)2-(YafQ)2 revealed that the N-terminal region of DinJ folds into a ribbon-helix-helix motif and dimerizes for DNA recognition, and the C-terminal portion of each DinJ exclusively wraps around a YafQ molecule. Upon incorporation into the heterotetrameric complex, a conformational change of YafQ in close proximity to the catalytic site of the typical microbial ribonuclease fold was observed and validated. Mutagenesis experiments revealed that a DinJ mutant restored YafQ RNase activity in a tetramer complex in vitro but not in vivo. An electrophoretic mobility shift assay showed that one of the palindromic sequences present in the upstream intergenic region of DinJ served as a binding sequences for both the DinJ-YafQ complex and the antitoxin DinJ alone. Based on structure-guided and site-directed mutagenesis of DinJ-YafQ, we showed that two pairs of amino acids in DinJ were important for DNA binding; the R8A and K16A substitutions and the S31A and R35A substitutions in DinJ abolished the DNA binding ability of the DinJ-YafQ complex.